Placental morphogenesis is critically important not only for pregnancy initiation, but also for the achievement of optimal placental function and an optimal environment for fetal growth. The mechanisms which control the characteristic organization of trophoblast and fetal villous placental vasculature during placental morphogenesis remain elusive, in part due to the inability to investigate differentiation at the level of the embryo and the implantation site during human pregnancy. Human embryonic stem cells (hESC) can differentiate to cells of the trophoblast lineage, and our preliminary studies show that cell-cell and cell-matrix interactions may play an important instructive role in trophoblast differentiation from hESC within embryoid bodies. Human ESC thus offer an exciting opportunity to address this critical developmental window at the cellular and molecular level. Our overall hypothesis is that placental morphogenesis requires the initiation of intrinsic pathways and response to extrinsic influences/cues to coordinate lineage determination, trophoblast differentiation and formation of the chorionic villi. We propose three Specific Aims to address this hypothesis. Specific Aim 1. To define trophoblast formation and differentiation in hESC directed to form embryoid bodies and exposed to varied extracellular matrix environments. Specific Aim 2. To evaluate trophoblast differentiation and morphogenesis in hESC/endothelial cell cocultures with 2D and 3D paradigms in vitro and in vivo. Specific Aim 3. To evaluate the effects of shear stress on phenotypic and flow-activated trophoblast functions. Few models exist for research in the areas of human embryonic development and implantation. Within the context of this P01 mechanism, interactions between trophoblasts and endothelial cells are hypothesized to contribute to differentiation as well as morphogenesis, and the study of trophoblast-enofof/?e//a/ interactions in two- and three-dimensional contexts and the impact of maternal vascular shear stress on differentiating trophoblasts could have major impact on our understanding of the control of human placental morphogenesis and pregnancy success.